Anti-inflammatory peptides and composition comprising the same

ABSTRACT

A peptide with anti-inflammatory activity, wherein the peptide comprises SEQ ID NO: 1, the peptide has above 80% homology of amino acid with above-mentioned sequence, or the peptide is the fragment of the above-mentioned peptides is described. An anti-inflammatory composition comprising the above mentioned peptides is described. According to the present invention, a peptide comprising a sequence of SEQ ID NO: 1 has outstanding efficacy in both suppressing inflammation and in prophylactic means. Therefore, the composition comprising the peptide of this invention can be used as anti-inflammatory pharmaceutical composition or as cosmetic composition, in turn, treating and preventing a variety of different types of inflammatory diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/343,502, filed on Nov. 4, 2016, which is a divisional applicationof U.S. application Ser. No. 14/400,291, filed Mar. 2, 2015, which is aU.S. National Stage of International Application No. PCT/EP2013/059460,filed May 7, 2013, said International Application No. PCT/EP2013/059460claims the benefit of Korean Application Nos. KR 10-2012-0104207 and KR10-2012-0104144 filed on Sep. 19, 2012; Korean Application No. KR10-2012-0071989 filed on Jul. 2, 2012; Korean Application Nos. KR10-2012-0050533 and KR 10-2012-0050529 filed on May 11, 2012; andInternational Application No. PCT/EP2013/055329 filed on Mar. 15, 2012,all of which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to anti-inflammatory peptides andcompositions comprising the same.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2473_0760007_Sequence listing ST25.txt; 3,284 bytes; and Date ofCreation: Sep. 11, 2018) was originally submitted in the InternationalApplication No. PCT/EP2013/059460 and is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Inflammation is a type of biological defense as a means of protectingthe body from damage of biological tissues that could be caused byexternal physical stimuli, chemical stimuli such as exposure to variousallergens, or invasion of microorganisms including bacteria, fungi andviruses.

The Cyclooxygenase (COX) pathway or Lipoxygenase (LOX) Pathway can usedfor signaling inflammation, which produce prostaglandin, thromboxane,etc. Once the inflammatory signal is delivered, one of many changes thathappen in the body is the expansion of the blood vessel for increasedblood supply around the inflammation to concentrate blood cells such asneutrophils required for the inflammatory response. However,inflammatory diseases can result if an abnormal biological defenseresponse occurs excessively. To prevent this, drugs that suppressexcessive inflammatory responses by repressing enzymes used ininflammatory signaling pathway (for example, COX-1, COX-2, 5-LOX, 12-LOXetc.) are under development.

According to response time, inflammation is categorized as acuteinflammation (immediate response, non-specific response, several days toseveral weeks), chronic inflammation (delayed response, specificresponse, several weeks or more), subacute inflammation (a middle stagein between acute inflammation and chronic inflammation, characteristicsof mixed product of mononuclear and polymorphounuclear).

Also, aside from peptide factors, factors such as prostaglandin,leukotriene, lipid factors including platelet activating factor (PAF),synthetic enzyme of inflammation factor, free radical such as NO (nitricoxide), many kinds of cell adhesion molecules, the immune system, andcoagulation factors can cause inflammation.

Once a cell is damaged due to the known causative agents of inflammationsuch as external biological factors (microbes, viruses, parasites),physical factors (mechanical stimuli, heat, radiation, electricity), andchemical factors, histamine and kinin are released. The releasedhistamine and kinin will result in angiectasis, increased capillarypermeability and concentration of macrophages at the inflammation site,and it causes increased blood flow rate, edema, immunocyte and antibodymigration, pain and heat generation.

Currently used treatments for inflammation are synthetic drugs such asibuprofen, antihistamines, steroids, cortisone, immunosuppressiveagents, and immune agonist; those which only temporarily alleviateinflammation. These drugs do not fundamentally cure inflammation, andthey have side effects such as hypersensitivity reaction, anddeterioration of immune system,

Therefore, for effective alleviation of inflammation, research isconducted to develop a substance that inhibits expression of the abovementioned inflammatory proteins. However, problems have arisen inanti-inflammation substances that had been developed previously. Diversecategories of anti-inflammatory drugs including Non-steroidalAnti-inflammatory Drugs (NSAIDs) and Steroidal Anti-inflammatory Drugs(SAIDs) have been developed; but not only do these drugs often bear sideeffects upon use, they also do not fundamentally cure the inflammation.Thus, there is a current need for anti-inflammatory drugs that are bothphysically and economically feasible. As one example, in acute orchronic inflammations such as chronic rheumatoid arthritis, not only donon-steroidal anti-inflammatory drugs suppress COX-2 enzyme activity,they are also known to suppress COX-1 activity, causing side effectssuch as gastrointestinal disorders.

The present invention was completed as present inventors have found thatpeptides derived from telomerase can have anti-inflammatory properties.

Therefore the objective of this invention is to provide a novel peptide.

Another objective of present invention is to provide the polynucleotidethat codes the novel peptide.

Another objective of present invention is to provide a peptide that hasanti-inflammatory activity.

Another objective of present invention is to provide ananti-inflammatory composition that uses this peptide as an activeingredient.

Another objective of present invention is to provide a cosmeticcomposition that uses this peptide as an active ingredient.

Another objective of present invention is to provide a pharmaceuticalcomposition that uses this peptide as an active ingredient.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, a peptide withanti-inflammatory activity, wherein the peptide comprises of amino acidsequence of SEQ ID NO: 1, or where the peptide has at least 80% homologywith the amino acid sequence with SEQ ID NO: 1, or the peptide is afragment of the above-mentioned peptides, is provided.

In another embodiment, the above-mentioned fragment consists of 3 ormore amino acids. For instance, the fragment may consist of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,or 26 amino acid residues.

In another embodiment, the above-mentioned peptide consists of 30 orless amino acids.

In another embodiment, the above-mentioned peptide consists of aminoacid sequence of SEQ ID NO: 1. For instance, the peptide may consist of29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, or 8 amino acid residues.

In another embodiment, the above-mentioned peptide originates from humantelomerase.

In one embodiment of the present invention, a polynucleotide encoding apeptide with anti-inflammatory activity, wherein the peptide comprisesamino acid sequence of SEQ ID NO: 1, or the peptide has at least 80%sequence identity with SEQ ID NO: 1, or the peptide is a fragment ofabove-mentioned peptides, is provided.

In another embodiment of the polynucleotide, the above-mentionedfragment is made of at least 3 amino acids. For instance, the fragmentmay consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or 26 amino acid residues.

In another embodiment of the polynucleotide, the above-mentioned peptideconsists of 30 or less amino acids. For instance, the peptide mayconsist of 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, or 8 amino acid residues.

In another embodiment of the polynucleotide, the above-mentioned peptideconsists of an amino acid sequence of SEQ ID NO: 1.

In another embodiment of the polynucleotide, the above-mentioned peptideoriginates from human telomerase.

In one embodiment of the present invention, anti-inflammatorycomposition comprising a peptide as active ingredient, wherein thepeptide comprises of amino acid sequence of SEQ ID NO: 1, the peptidehas above 80% homology of amino acid sequence with SEQ ID NO: 1, or thepeptide is a fragment of the above-mentioned peptides, is provided.

In another embodiment of the composition, the above-mentioned peptideconsists of at least 3 amino acids, cf. above.

In another embodiment of the composition, the above-mentioned peptideconsists of 30 or less amino acids, cf. above.

In another embodiment of the composition, the above-mentioned peptideconsists of an amino acid sequence of SEQ ID NO: 1.

In another embodiment of the composition, the above-mentioned peptideoriginates from human telomerase.

In another embodiment of the composition, the above-mentionedcomposition is for treatment or prophylaxis of inflammatory disease.

In another embodiment of the composition, the above-mentionedcomposition is a cosmetic composition for improving or preventing skininflammation.

In another embodiment of the composition, the above-mentionedcomposition is a pharmaceutical composition for treatment or prophylaxisof inflammatory disease.

In another embodiment of the composition, the above-mentionedcomposition is a food composition for treatment or prophylaxis ofinflammation.

In another embodiment of the composition, the above-mentionedinflammatory disease is characterized by selecting from the groupconsisting of (1) general or localized inflammatory disease (forexample, allergies; immune-complex disease; hayfever; hypersensitiveshock; endotoxin shock; cachexia, hyperthermia; granulomatosis; orsarcoidosis); (2) gastrointestinal related diseases (for example,appendicitis; gastric ulcer; duodenal ulcer; peritonitis; pancreatitis;ulcerative, acute, or ischemic colitis; cholangitis; cholecystitis,steatorrhea, hepatitis, Crone's disease; or Whipple's Disease); (3)dermal related diseases (for example, psoriasis; burns; sunburns;dermatitis; Urticarial warts or wheal); (4) vascular related diseases(for example, angiitis; vasculitis; endocarditis; arteritis;atherosclerosis; thrombophlebitis; pericarditis; congestive heartfailure; myocarditis; myocardial ischemia; periarteritis nodosa;recurrent stenosis; Buerger's disease; or rheumatic fever); (5)respiratory diseases (for example, asthma; epiglottitis; bronchitis;emphysema; rhinitis; cystic fibrosis; interstitial pneumonitis; COPD(chronic obstructive pulmonary disease); adult respiratory distresssyndrome; coniosis; alveolitis; bronchiolitis; pharyngitis; pleurisy; orsinusitis); (6) bone, joint, muscle and connective tissue relateddiseases (for example, eosinophilic granuloma; arthritis; arthralgia;osteomyelitis; dermatomyositis; fasciitis; Paget's disease; gout;periodontal disease; rheumatoid arthritis; myasthenia gravis; ankylosingspondylitis; or synovitis); (7) urogenital disorders (for example,epididymitis; vaginitis; prostatitis; or urethritis); (8) central orperipheral nervous system related diseases (for example, Alzheimer'sdisease; meningitis; encephalitis; multiple sclerosis; cerebralinfarction; cerebral embolism; Guillain-Barre syndrome; neuritis;neuralgia; spinal cord injury; paralysis; or uveitis); (9) virus (forexample, influenza; respiratory syncytial virus; HIV; hepatitis B;hepatitis C; or herpes virus), infectious disease (for example, Denguefever; or septicemia), fungal infection (for example, candidiasis); orbacterial, parasitic, and similar microbial infection (for example,disseminated bacteremia; malaria; onchocerciasis; or amebiasis); (10)autoimmune disease (for example, thyroiditis; lupus; Goodpasture'ssyndrome; allograft rejection; graft versus host disease; or diabetes);and (11) cancer or tumor disease (for example, Hodgkin's disease).

In one embodiment of the present invention, a method for treating orpreventing inflammatory diseases by administering the anti-inflammatorycomposition is provided.

In one embodiment of the present invention, a kit for prophylaxis ortreatment of inflammatory disease comprising: a peptide withanti-inflammatory activity or a composition comprising the peptide,wherein the peptide comprises amino acid sequence of SEQ ID NO: 1, thepeptide has above 80% amino acid sequence homology with SEQ ID NO: 1, orthe peptide is a fragment of above-mentioned peptides; and instructionsincluding at least one of administration dose, administration route,administration frequency, and indication of the peptide or composition,is provided.

INDUSTRIAL APPLICABILITY

According to the present invention, a peptide that has a sequence of SEQID NO: 1 has outstanding efficacy in both suppressing inflammation andin prophylactic means. Therefore, the composition comprising thepeptides of this invention can be used as anti-inflammatorypharmaceutical composition or as cosmetic composition, in turn, treatingand preventing a variety of different types of inflammatory diseases.

REFERENCE

-   KR2012-0130996A-   KR2012-0133661A-   KR2011-0060940A-   US2011-0150873A1-   Bonaldi T et al., EMBO J, (22)5551-60, 2003-   Yankner B A et al, Science (New York, N.Y.) [1990, 250(4978):    279-282]-   Dahlgren K N et al, J. Biol. Chem. 277: 32046-32053, 2002.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows the results of performing TNF-α ELISA withthe culture of monocytes derived from PBMC. The monocytes werestimulated with LPS (10 ng/ml) for two hours, then reacted with eachpeptide, FITC, FITC-TAT, PEP 1-FITC and FITC-peptide for two hours.(**P<0.01. Compared with the negative control (FITC and FITC-TAT).

FIG. 2 is a graph which shows the results of performing luciferaseanalysis from transfecting HEK293/null and HEK293/TLR2 cell lines withNF-κB luciferase, then reacting with lipoprotein (10 ng/ml) and FITC andFITC-PEP-1 (4 μM), and incubating for 18 hours. Results of luciferasewere obtained by correction using renilla. (**P<0.01. compared to thenegative control (No treat) and compared with lipoprotein treatedsample.)

FIG. 3 represents the inhibition levels of cytokines in THP1 cell linewhich were treated with none, LPS, PEP 1 and LPS+PEP 1.

FIG. 4 represents viability of neural stem cell treated with 0, 2.5,5.0, 10, 20 and 40 μM of amyloid-β protein.

FIG. 5 represents proliferation of neural stem cell treated with 0, 2.5,5.0, 10, 20 and 40 μM amyloid-β protein.

FIG. 6 represents viability of neural stem cell treated with 0, 1, 10,50, 100 and 200 μM of PEP 1.

FIG. 7 represents proliferation of neural stem cell treated with 0, 1,10, 50, 100 and 200 μM of PEP 1.

FIG. 8 represents viability of neural stem cell treated with 1, 10, 50and 100 μM PEP 1; neural stem cells were damaged by 20 μM of amyloidbeta protein, and then cell viability was measured after treatment withdifferent concentrations of PEP-1. (Control groups were those untreatedwith amyloid beta protein and telomerase-based peptides).

FIG. 9 represents toxicity of neural stem cell treated with 1, 10, 50and 100 μM PEP 1; neural stem cells were damaged by 20 μM of amyloidbeta protein, and then cell toxicity was measured after treatment withdifferent concentrations of PEP-1. (Control groups were those untreatedwith amyloid beta protein and telomerase-based peptides).

FIG. 10 represents proliferation of neural stem cells treated with 1,10, 50 and 100 μM PEP 1; neural stem cells were damaged by 20 μM ofamyloid beta protein, and then cell proliferation was measured aftertreatment with different concentrations of PEP-1. (Control groups werethose untreated with amyloid beta protein and telomerase-basedpeptides).

FIG. 11 represents migration of neural stem cells treated with 1, 10, 50and 100 μM PEP 1; neural stem cells were damaged by 20 μM of amyloidbeta protein, and then cell migration was measured after treatment withdifferent concentrations of PEP-1. (Control groups were those untreatedwith amyloid beta protein and PEP-1).

FIG. 12 represents apoptosis of neural stem cells treated with 1, 10, 50and 100 μM PEP 1; neural stem cells were damaged by 20 μM of amyloidbeta protein, and then cell apoptosis was measured after treatment withdifferent concentrations of PEP-1. (Control groups were those untreatedwith amyloid beta protein and telomerase-based peptides).

FIG. 13 represents ROS (Reactive Oxygen Species) inhibitory effect ofPEP-1 in neural stem cells damaged by amyloid beta peptide; neural stemcells were damaged by 20 μM of amyloid beta protein, and then inhibitionof ROS was measured after treatment with different concentrations ofPEP-1 (1, 10, 50 and 100 μM). (Control groups were those untreated withamyloid beta protein and PEP-1).

FIG. 14 represents the results of protein expression levels analyzed by(A) 2D-eletrophoresis and (B) Antibody Array; neural stem cells weredamaged by 20 μM of amyloid beta protein, and then protein expressionlevel was measured after treatment with different concentrations ofPEP-1 (1, 10 and 50 μM). (Control groups were those untreated withamyloid beta protein and PEP-1).

FIG. 15 represents the results of western blot showing the expressionlevel of inflammation-related proteins: neural stem cells were damagedby 20 μM of amyloid beta protein, and then cells were treated withdifferent concentrations of PEP-1 (1, 10 and 50 μM).

FIG. 16 represents the inhibitory effect of PEP 1 on amyloid betaprotein aggregation; (A) shows the reduced oligomerization of amyloidbeta proteins when co-treated with 1 μM amyloid beta protein and PEP 1(0.1, 1 and 10 μM). (B) shows the case when PEP-1 was treated on theamyloid-β protein that was already induced for aggregation.

FIG. 17 represents the effect of PI3K-inhibitor, LY294002 on the cellviability treated with PEP 1. The increased cell viability aftertreating with PEP 1 decreased when treated with LY294002.

DETAILED DESCRIPTION OF THE INVENTION

Since the present invention can have adaptability for diversetransformation and examples of practical application, below is a moredetailed description of the present invention. Nevertheless, this is nomeans to limit the form of practical application; it should beunderstood that the intention is to include the concept and the extentof technology in all of the transformation, equivalents to alternatives.In describing the present invention, if any detailed description aboutthe prior art is considered to deteriorate the fundamental principles ofthe present invention, the description will be omitted.

A telomere is known as a repetitive sequence of genetic material at theends of chromosomes that prevent chromosomes from damage or merging ofother chromosomes. The length of a telomere is shortened at each celldivision, and after a certain number of cell division, the telomerelength is extremely shortened to the extent in which the cell stopsdividing and dies. On the other hand, the elongation of telomeres isknown to extend the life span of a cell. For an example, cancer cellsexcrete an enzyme called telomerase, which prevents shortening oftelomeres, thus resulting in proliferation of cancer cells. The presentinvention was accomplished upon the discovery of telomerase-derivedpeptides with anti-inflammatory effects.

In one embodiment of the present invention, a peptide withanti-inflammatory activities is provided. The peptide comprises at leastone amino acid sequence of SEQ ID NO: 1, the peptide has above 80%homology with above-mentioned sequence, or the peptide is a fragment ofthe above-mentioned peptides.

Peptide with anti-inflammatory activity in the present invention is apeptide having an amino acid sequence to SEQ ID NO: 1. Peptide of SEQ IDNO: 1 is a peptide consisting of 16 amino acids in the location oftelomerase-[611-626].

SEQ ID NO: 1 EARPALLTSRLRFIPK

In one embodiment of the present invention, a polynucleotide that codesa peptide with anti-inflammatory activities is provided. Thepolynucleotide codes a peptide comprising at least one amino acidsequence of SEQ ID NO: 1, a peptide having above 80% homology withabove-mentioned sequence, or a peptide being a fragment of theabove-mentioned peptides. The polynucleotide mentioned above enablesproduction of the peptides in large quantities. For example, cultivationof vectors that include polynucleotides encoding peptides allowsproduction of peptides in large quantities.

The peptides disclosed herein can include a peptide comprising aminoacid sequence above 80%, above 85%, above 90%, above 95%, above 96%,above 97%, above 98%, or above 99% homology. Moreover, the peptidesdisclosed in the present invention can include a peptide comprising SEQID NO: 1 or its fragments, and a peptide with more than 1 transformedamino acid, more than 2 transformed amino acid, more than 3 transformedamino acid, more than 4 transformed amino acid, more than 5 transformedamino acid, more than 6 transformed amino acid, or more than 7transformed amino acid.

In the present specification and claims, the terms “homology” and“sequence identity” are used interchangeably to indicate the degree ofsequence overlap between two amino acid (or if relevant: nucleic acid)sequences.

Unless otherwise stated the term “Sequence identity” for peptides asused herein refers to the sequence identity calculated as(n_(ref)−n_(dif))−100/n_(ref), wherein n_(dif) is the total number ofnon-identical residues in the two sequences when aligned so that amaximum number of amino acids are identical and wherein n_(ref) is thenumber of residues in the shortest of the sequences. Hence, the DNAsequence agtcagtc will have a sequence identity of 75% with the sequenceaatcaatc (n_(dif)=2 and n_(ref)=8).

In some embodiments the sequence identity is determined by conventionalmethods, e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by thesearch for similarity method of Pearson & Lipman, 1988, Proc. Natl.Acad. Sci. USA 85: 2444, using the CLUSTAL W algorithm of Thompson etal., 1994, Nucleic Acids Res 22:467380, by computerized implementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group). The BLAST algorithm(Altschul et al., 1990, Mol. Biol. 215:403-10) for which software may beobtained through the National Center for Biotechnology Informationwww.ncbi.nlm.nih.gov/) may also be used. When using any of theaforementioned algorithms, the default parameters for “Window” length,gap penalty, etc., are used.

In one embodiment of the present invention, changes in amino acidsequence belong to the modification of peptide's physical and chemicalcharacteristics. For example, amino acid transformation can be performedby improving thermal stability of the peptide, altering substratespecificity, and changing the optimal pH.

In one embodiment of the present invention, a peptide comprising aminoacid sequence of SEQ ID NO: 1, a peptide comprising of amino acidsequence above 80% homology with above-mentioned sequence or a peptidefragment of above-mentioned peptides is preferably made of 30 or lessamino acids.

In one embodiment of the present invention, a peptide comprising aminoacid sequence of SEQ ID NO: 1, a peptide comprising of amino acidsequence above 80% homology with above-mentioned sequence or a peptidefragment of above-mentioned peptides comprises a peptide originates fromtelomerase, more specifically, telomerase of Homo sapiens.

The term “amino acid” herein includes not only the 22 standard aminoacids that are naturally integrated into peptide but also the D-isomersand transformed amino acids. Therefore, in a specific embodiment of thepresent invention, a peptide herein includes a peptide having D-aminoacids. On the other hand, a peptide may include non-standard amino acidssuch as those that have been post-translationally modified. Examples ofpost-translational modification include phosphorylation, glycosylation,acylation (including acetylation, myristorylation, plamitoylation),alkylation, carboxylation, hydroxylation, glycation, biotinylation,ubiquitinylation, transformation in chemical properties (e.g. β-removingdeimidation, deamidation) and structural transformation (e.g. formationof disulfide bridge). Also, changes of amino acids are included and thechanges of amino acids occur due to chemical reaction during thecombination process with crosslinkers for formation of a peptideconjugate.

A peptide disclosed herein may be a wild-type peptide that has beenidentified and isolated from natural sources. On the other hand, whencompared to peptide fragments of SEQ ID NO: 1, the peptides disclosedherein may be artificial mutants that comprise one or more substituted,deleted and/or inserted amino acids. Amino acid alteration in wild-typepolypeptide—not only in artificial mutants—comprises conservativesubstitution of amino acids that do not influence protein folding and oractivation. Examples of conservative substitution belong to the groupconsisting of basic amino acids (arginine, lysine and histidine), acidicamino acids (glutamic acid and aspartic acid), polar amino acids(glutamine and asparagines), hydrophobic amino acids (leucine,isoleucine, valine and methionine), aromatic amino acids (phenylalanine,tryptophan and tyrosine), and small amino acids (glycine, alanine,serine, and threonine). The amino acid substitutions that do notgenerally alter the specific activity are known in the art of thepresent invention. Most common occurred alteration are Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and theopposite alterations. Another example of conservative substitutions areshown in the following table 1.

TABLE 1 Original Preferable residue amino acid Examples of residuesubstitution substitution Ala (A) val; leu; ile Val Arg (R) lys; gln;asn Lys Asn (N) gln; his; asp, lys; arg Gln Asp (D) glu; asn Glu Cys (C)ser; ala Ser Gin (Q) asn; glu Asn Glu (E) asp; gln Asp Gly (G) ala AlaHis (H) asn; gln; lys; arg Arg Ile (I) leu; val; met; ala; phe;norleucine Leu Leu (L) norleucine; ile; val; met; ala; phe Ile Lys (K)arg; gln; asn Arg Met (M) leu; phe; ile Leu Phe (F) leu; val; ile; ala;tyr Tyr Pro (P) ala Ala Ser (S) thr Thr Thr (T) ser Ser Trp (W) tyr; pheTyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; ala;norleucine Leu

The substantial transformation of the biological properties of peptidesare performed by selecting significantly different substitution in thefollowing efficacies: (a) the efficacy in maintaining the structure ofthe polypeptide backbone in the area of substitution, such as sheet orhelical three-dimensional structures, (b) the efficacy in maintainingelectrical charge or hydrophobicity of the molecule in the target area,or (c) the efficacy of maintaining the bulk of the side chain. Naturalresidues are divided into groups by general side chain properties as thefollowing:

(1) hydrophobicity: Norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilicity: cys, ser, thr;

(3) acidity: asp, glu;

(4) basicity: asn, gln, his, lys arg;

(5) residue that affects chain orientation: gly, pro; and

(6) aromaticity: trp, tyr, phe.

Non-conservative substitutions may be performed by exchanging a memberof the above classes to a different class's. Any cysteine residues thatare not related in maintaining the proper three-dimensional structure ofthe peptide can typically be substituted into serine, thus increasingthe oxidative stability of the molecule and preventing impropercrosslinkage. Conversely, improvement of stability can be achieved byadding cysteine bond(s) to the peptide.

Altered types of amino acids variants of peptides are those thatantibody glycosylation pattern changed. The term “change” herein relatesto deletion of carbohydrate residues and/or addition of at least oneglycosylated residues that do not exist within a peptide.

Glycosylation in peptides are typically N-connected or O-connected. Theterm “N-connected” herein relates to that carbohydrate residues areattached to the side chain of asparagine residues. As tripeptidesequences, asparagine-X-serine and asparagine-X-threonine (where the Xis any amino acid except proline) are the recognition sequence forattaching carbohydrate residue enzymatically to the side chain ofasparagine. Therefore, with the presence of one of these tripeptidesequences in a polypeptide, the potential glycosylation sites arecreated. “O-connected glycosylation” means attaching one of sugarN-acetylgalactosamine, galactose, or xylose to hydroxyl amino acids. Thehydroxyl amino acids are most typically serine or threonine, but5-hydroxyproline or 5-hydroxylysine can be used.

Addition of glycosylation site to a peptide is conveniently performed bychanging amino acid sequence to contain tripeptide sequence mentionedabove (for N-linked glycosylation sites). These changes may be made byaddition of at least one serine or theronine residues to the firstantibody sequence, or by substitution with those residues (for O-linkedglycosylation sites).

In one embodiment of the present invention, a polynucleotide is anucleic acid molecule that can be spontaneous or artificial DNA or RNAmolecules, either single-stranded or double-stranded. The nucleic acidmolecule can be one or more nucleic acids of same type (for example,having a same nucleotide sequence) or nucleic acids of different types.The nucleic acid molecules comprise one or more DNA, cDNA, decoy DNA,RNA, siRNA, miRNA shRNA, stRNA, snoRNA, snRNA PNA, antisense oligomer,plasmid and other modified nucleic acids, but not limited to those.

A HMGB1 protein is known as a cytokine. It first undergoes acetylationand translocation to cytoplasm by external stimulation. Then it issecreted out of the cell, therefore serving the role ofinflammation-causing cytokine. Because when one has an inflammation dueto such activity, HMGB1 protein is secreted out of the cell, andpatients with inflammatory diseases such as Churg strauss syndrome,rheumatoid arthritis and Sjogren's syndrome will present with elevatedserum levels of HMGB1. Hence, if nucleus contains large amount of HMGB1even when there is a stimulus that causes inflammation, it is suggestiveof the fact that HMGB1 is not being secreted out of the cell, whichmeans inflammation is being suppressed.

In one embodiment of the present invention, when treated a cell with apeptide comprising amino acid sequence of SEQ ID NO: 1, a peptide havingabove 80% homology of amino acid sequence with above-mentioned sequence,or a fragment of the above-mentioned peptides, amount of HMGB1 withinthe nucleus increases. This represents that the peptides mentioned abovehave excellent inflammation preventive or suppressive effects.

Also, in specific embodiments of the present invention, a peptidecomprising amino acid sequence of SEQ ID NO: 1, a peptide having above80% homology of amino acid sequence with above-mentioned sequence, or afragment of the above-mentioned peptides, has an advantage in that ithas high feasibility due to its low toxicity within a cell.

In the present invention, an “inflammatory disease” is a broadindication that refers to any disease that designates inflammation as amain cause or inflammation caused by disease. Specifically, aninflammatory disease includes (1) general or localized inflammatorydisease (for example, allergies; immune-complex disease; hayfever;hypersensitive shock; endotoxin shock; cachexia, hyperthermia;granulomatosis; or sarcoidosis); (2) gastro-intestinal related diseases(for example, appendicitis; gastric ulcer; duodenal ulcer; peritonitis;pancreatitis; ulcerative, acute, or ischemic colitis; cholangitis;cholecystitis, steatorrhea, hepatitis, Crone's disease; or Whipple'sDisease); (3) dermal related diseases (for example, psoriasis; burns;sunburns; dermatitis; Urticarial warts or wheal); (4) vascular relateddiseases (for example, angiitis; vasculitis; endocarditis; arteritis;atherosclerosis; thrombophlebitis; pericarditis; congestive heartfailure; myocarditis; myocardial ischemia; periarteritis nodosa;recurrent stenosis; Buerger's disease; or rheumatic fever); (5)respiratory diseases (for example, asthma; epiglottitis; bronchitis;emphysema; rhinitis; cystic fibrosis; interstitial pneumonitis; COPD(chronic obstructive pulmonary disease); adult respiratory distresssyndrome; coniosis; alveolitis; bronchiolitis; pharyngitis; pleurisy; orsinusitis); (6) bone, joint, muscle and connective tissue relateddiseases (for example, eosinophilic granuloma; arthritis; arthralgia;osteomyelitis; dermatomyositis; fasciitis; Paget's disease; gout;periodontal disease; rheumatoid arthritis; myasthenia gravis; ankylosingspondylitis; or synovitis); (7) urogenital disorders (for example,epididymitis; vaginitis; prostatitis; or urethritis); (8) central orperipheral nervous system related diseases (for example, Alzheimer'sdisease; meningitis; encephalitis; multiple sclerosis; cerebralinfarction; cerebral embolism; Guillain-Barre syndrome; neuritis;neuralgia; spinal cord injury; paralysis; or uveitis); (9) virus (forexample, influenza; respiratory syncytial virus; HIV; hepatitis B;hepatitis C; or herpes virus), infectious disease (for example, Denguefever; or septicemia), fungal infection (for example, candidiasis); orbacterial, parasitic, and similar microbial infection (for example,disseminated bacteremia; malaria; onchocerciasis; or amebiasis); (10)autoimmune disease (for example, thyroiditis; lupus; Goodpasture'ssyndrome; allograft rejection; graft versus host disease; or diabetes);and (11) cancer or tumor disease (for example, Hodgkin's disease), butnot limited to those.

Treating the inflammatory component of such diseases has been a majorgoal of the global pharmaceutical industry for a number of decades, anda wide variety of useful treatments have been developed. Examplesinclude the corticosteroids (a range of natural, semisynthetic andsynthetic agents designed to mimic the effect of Cortisol, includingprednisolone, methylprednisolone, dexamethasone, betamethasone,fluticasone and so forth), cyclooxygenase inhibitors (both non-selectiveor cox-1 selective, such as indomethacin, sulfasalzine and aspirin, andmore recently cox-2 selective, such as celecoxib), leukotriene blockers(such as monteleukast) and anti-TNFs (such as modified monoclonalneutralising antibodies, including infliximab (Remicade™) and adalimumab(Humira™), TNF receptor fusion proteins, such as etanercept (Enbrel™),as well as small molecule TNF-α synthesis inhibitors like thalidomide).

In one embodiment of the present invention, an anti-inflammatorycomposition comprising a peptide as an active ingredient is provided.The peptide comprises amino acid sequence of SEQ ID NO: 1, the peptidehas above 80% homology with above-mentioned sequence, or the peptide isa fragment of the above-mentioned peptides.

In one embodiment of the present invention, the anti-inflammatorycomposition may contain 0.1 μg/mg to 1 mg/mg, specifically 1 μg/mg to0.5 mg/mg, more specifically 10 μg/mg to 0.1 mg/mg of a peptidecomprising of amino acid sequence SEQ ID NO: 1, a peptide comprising ofamino acid sequence above 80% homology with above-mentioned sequence, orpeptide fragment of above-mentioned peptides. When the peptide iscontained in the above mentioned range, all the safety and stability ofthe composition may be satisfied and appropriate in terms ofcost-effectiveness.

In one embodiment of the present invention, the composition may haveapplication with all animals including human, dog, chicken, pig, cow,sheep, guinea pig, and monkey.

In one embodiment of the present invention, the pharmaceuticalcomposition for the use of treatment or prophylaxis of inflammatorydisease with an active ingredient that is comprised of a peptideconsisting of an amino acid of SEQ ID NO: 1, a peptide comprising ofamino acid sequence above 80% homology with above-mentioned sequence, orpeptide fragment of SEQ ID NO: 1, is provided. In one embodiment of thepresent invention, the pharmaceutical composition may be administeredthrough oral, rectal, transdermal, intravenous, intramuscular,intraperitoneal, in the bone marrow, epidural or subcutaneous means.

Forms of oral administration may be, but not limited to, tablets, pills,soft or hard capsules, granules, powders, solution, or emulsion. Formsof non-oral administration may be, but not limited to, injections,drips, lotions, ointments, gels, creams, suspensions, emulsions,suppository, patch, or spray.

In one embodiment of the present invention, the pharmaceuticalcomposition, if necessary, may contain additives, such as diluents,excipients, lubricants, binders, disintegrants, buffers, dispersants,surfactants, coloring agents, aromatics or sweeteners. In one embodimentof the present invention, the pharmaceutical composition can bemanufactured by conventional methods of the industry in the art.

In one embodiment of the present invention, the active ingredient of thepharmaceutical composition may vary according to the patient's age, sex,weight, pathology and state, administration route, or prescriber'sjudgment. Dosage based on these factors is determined within levels ofthose skilled in the art, and the daily dose for example may be, but notlimited to, 0.1 μg/kg/day to 1 g/kg/day, specifically 1 μg/kg/day to 10mg/kg/day, more specifically the 10 μg/kg/day to 1 mg/kg/day, morespecifically the 50 μg/kg/day to 100 μg/kg/day. In one embodiment of thepresent invention, the pharmaceutical composition may be administered,but not limited to, 1 to 3 times a day.

In one embodiment of the present invention, a skin external compositionfor improvement or prevention of skin inflammation is provided. The skinexternal composition may contain an active ingredient that is a peptidecomprising of an amino acid sequence of SEQ ID NO: 1, a peptidecomprising of amino acid sequence above 80% homology withabove-mentioned sequence, or a peptide fragment of above-mentionedpeptides.

In another embodiment of the present invention, a cosmetic compositionfor improvement or prevention of skin inflammation is provided. Thecosmetic composition may contain an active ingredient that is a peptidecomprising of an amino acid sequence of SEQ ID NO: 1, a peptidecomprising of amino acid sequence above 80% homology withabove-mentioned sequence, or peptide fragment of above-mentionedpeptides.

In one embodiment of the present invention, external applicationcomposition or cosmetic composition may be provided in all formsappropriate for topical applications. For example, forms can be providedas solutions, emulsions obtained by dispersion of oil phase in water,emulsion obtained by dispersion of water in oil phase, suspension,solid, gel, powder, paste, foam or aerosol. These forms can bemanufactured by conventional methods of the industry in the art.

In one embodiment of the present invention, the cosmetic composition mayinclude, within levels that will not harm the main effect, otheringredients that can desirably increase the main effect. In oneembodiment of the present invention, the cosmetic composition mayadditionally include moisturizer, emollient agents, surfactants, UVabsorbers, preservatives, fungicides, antioxidants, pH adjusting agent,organic or inorganic pigments, aromatics, cooling agent orantiperspirant. The formulation ratio of the above-mentioned ingredientscan be decided by those skilled in the art within levels that will notharm the purpose and the effects of the present invention, and theformulation ratio based on total weight of the cosmetic composition canbe 0.01 to 5% by weight, specifically 0.01 to 3% by weight.

In one embodiment of the present invention, a food composition forinflammation prevention or suppression is provided. The food compositionmay contain with an active ingredient that is a peptide comprising of anamino acid sequence of SEQ ID NO: 1, a peptide comprising of amino acidsequence above 80% homology with above-mentioned sequence, or peptidefragment of above-mentioned peptides.

In one embodiment of the present invention, food composition is notlimited to forms, but for example may be granules, powder, liquid, andsolid forms. Each form can be formed with ingredients commonly used inthe industry appropriately chosen by those skilled in the art, inaddition to the active ingredient, and can increase the effect withother ingredients.

Decision for dosage on the above-mentioned active ingredient is withinthe level of those skilled in the art, and daily dosage for example maybe 1 μg/kg/day to 10 mg/kg/day, more specifically the 10 μg/kg/day to 1mg/kg/day, more specifically the 50 μg/kg/day to 100 μg/kg/day, but notlimited to these numbers and can vary according to age, health status,complications and other various factors.

In one embodiment of the present invention, a use of prevention ortreatment of inflammatory disease with a peptide comprising of an aminoacid sequence of SEQ ID NO: 1, a peptide comprising of amino acidsequence above 80% homology with above-mentioned sequence, or peptidefragment of above-mentioned peptides, is provided.

In one embodiment of the present invention, the method of prevention ortreatment of inflammatory disease with applying peptides mentioned abovein patients is provided.

In one embodiment of the present invention, a kit for prophylaxis ortreatment of inflammatory diseases is provided. The kit may contain: apeptide with anti-inflammatory activity or a composition comprising ofthe peptide, wherein the peptide comprises amino acid sequence of SEQ IDNO: 1, the peptide has above 80% homology with above-mentioned sequence,or the peptide is a fragment of the above-mentioned peptides; andinstructions including at least one of administration dose,administration route, administration frequency, and indication of thepeptide or composition.

The terms used herein is intended to be used to describe theembodiments, not to limit the present invention. Terms without numbersin front are not to limit the quantity but to show that there may bemore than one thing of the term used. The term “including”, “having”,“consisting”, and “comprising” shall be interpreted openly (i.e.“including but not limited to”).

Mention of range of numbers is used instead of stating separate numberswithin the range, so unless it is explicitly stated, each number can beread as separate numbers integrated herein. The end values of all rangesare included in the range and can be combined independently.

Unless otherwise noted or clearly contradicting in context, all methodsmentioned herein can be performed in the proper order. The use of anyone embodiment and all embodiment, or exemplary language (e.g., that use“like˜”), unless included in the claims, is used to more clearlydescribe the present invention, not to limit the scope of the presentinvention. Any language herein outside of the claims should not beinterpreted as a necessity of the present invention. Unless definedotherwise, technical and scientific terms used herein have meaningnormally understood by a person skilled in the art that the presentinvention belongs to.

The preferred embodiments of the present invention are the best modeknown to the inventors to perform the present invention. It may becomeclear to those skilled in the art after reading the statements ahead ofthe variations in the preferred embodiments. The present inventors hopethat those skilled in the art can use the variations adequately andpresent invention be conducted in other ways than listed herein. Thus,the present invention, as allowed by the patent law, includesequivalents, and variations thereof, of the key points of the inventionstated in the appended claims. In addition, all possible variationswithin any combination of the above-mentioned components are included inthe present invention, unless explicitly stated otherwise orcontradicting in context. Although the present invention is describedand shown by exemplary embodiments, those skilled in the art willunderstand well that there can be various changes in the form anddetails without departing from the spirit of the invention and range,defined by the claims below.

Tumor necrosis factor (TNF), particularly TNF-α, is known to be releasedfrom inflammatory cells and cause various cytotoxic reactions,immunological reactions and inflammatory reactions. TNF-α is known to beinvolved in the occurrence and prolongation of many inflammatory andautoimmune diseases and further cause serious septicemia and septicshock when it is released into the blood and acts systemically. BecauseTNF-α is a factor associated widely with the immune system of a livingbody, the development of agents inhibiting TNF-α is actively carriedout. TNF-α is biosynthesized in an inactive form and becomes an activeform by being cleaved by protease; the enzyme responsible for theactivation is called a tumor necrosis factor-converting enzyme (TACE).Thus, a substance inhibiting this TACE can treat, improve, or preventdiseases, pathologic conditions, abnormal conditions, troubles, adversesymptoms and the like ascribed to TNF-α (KR2011-0060940A).

High-mobility group box 1 (HMGB1) protein exists in high concentrationsin thymus, lymph nodes, testes, and in fetal liver, and with exceptionto liver and brain cells, usually exists inside of the nucleus. The saidHMGB1 protein has 3 domains consisting of A-box, B-box, and C-terminal.

It was reported by Tracey et al., 1999 that HMGB1 protein has a role asa cytokine which induces inflammation, and the mechanism of said HMGB1'sinflammation induction is by an external stimulus causing acetylation ofHMGB1 which then moves from the nucleus into the cytoplasm. Afterward,it is known to be secreted out of the cell, or secreted out from thecell in necrosis. (Bonaldi T et al., EMBO J, (22)5551-60, 2003).

The invention is further described by the figures, the followingexamples and experiments, which are solely for the purpose ofillustrating specific embodiments of this invention, and are not to beconstrued as limiting the scope of the invention in any way.

Example 1 Synthesis of PEP-1 and Measurement of Anti-InflammatoryActivities of PEP-1 (SEQ ID NO: 1)

Experiment 1-1: Synthesis of PEP-1 (SEQ ID NO: 1)

A peptide comprised of 16 amino acids with the chemical structure 1 asbelow having the sequence SEQ ID: 1 (PEP-1) derived from humantelomerase was synthesized:

-   -   SEQ ID NO: 1 (PEP-1) was synthesized according to the existing        method of solid phase peptide synthesis. In detail, the peptides        were synthesized by coupling each amino acid from C-terminus        through Fmoc solid phase peptide synthesis, SPPS, using ASP48S        (Peptron, Inc., Daejeon ROK). Those peptides with their first        amino acid at the C-terminus being attached to resin were used        as follows:

NH₂-Lys(Boc)-2-chloro-Trityl Resin

NH₂-Ala-2-chloro-Trityl Resin

NH₂-Arg(Pbf)-2-chloro-Trityl Resin

All the amino acid materials to synthesize the peptide were protected byFmoc at the N-terminus, and the amino acid residues were protected byTrt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl) that can be dissolved in acid. Such as:Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH,Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH,Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ahx-OH,Trt-Mercaptoacetic acid.

HBTU[2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminiumhexafluorophosphate]/HOBt [N-Hydroxybenzotriazole]/NMM[4-Methylmorpholine] were used as the coupling reagents. In 20% of DMF,piperidine was used to remove Fmoc. In order to remove the protectionfrom residue or to separate the synthesized peptide from Resin, cleavagecocktail [TFA (trifluoroacetic acid)/TIS (triisopropylsilane)/EDT(ethanedithiol)/H₂O=92.5/2.5/2.5/2.5] was used.

Synthesized the peptide by using the solid phase scaffold combined tostarting amino acid with the amino acid protection, reacting thecorresponding amino acids separately, washing with solvent anddeprotected, and repeating the process. After cutting off thesynthesized peptide from the resin, it was purified by HPLC and verifyfor synthesis by MS, and then freeze-dried.

Specific synthesis process of PEP 1 is described by the following.

-   -   1) Coupling

Melted the amino acid (8 equivalent) protected withNH₂-Lys(Boc)-2-chloro-Trityl Resin, and coupling agent HBTU (8equiv.)/HOBt (8 equiv.)/NMM (16 equiv.) and added to DMF, then let reactin room temperature for 2 hours, then washed with DMF, MeOH, and DMF inthat order.

-   -   2) Fmoc deprotection

Added 20% piperidine in DMF and reacted in room temperature for 5minutes 2 times, then washed with DMF, MeOH, and DMF in that order.

-   -   3) Make the basic framework of peptide by repeating reactions 1        and 2 repeatedly.    -   4) Cleavage: Add Cleavage Cocktail to the completely synthesized        peptide and separated the peptide from the resin.    -   5) Add cooling diethyl ether into obtained mixture, and then        centrifugation is used to precipitate gathered peptide.    -   6) After purification by Prep-HPLC, check the molecular weight        by LC/MS and lyophilize to produce in powder form.

Example 2 Anti-Inflammatory Activity Measurement of PEP-1

Cell Lines Culture

Raw 264.7macrophage cell (KCBL, 40071) from Korea Cell Bank wasmaintained in Dulbecco's modified Eagle's medium (DMEM; PAA, Austria)containing 10% fetal bovine serum (FBS; Gibco Laboratories), 100 unit/mLof streptomycin, and penicillin (Gibco Laboratories) at 37° C. with 5%CO. Raw264.7 cells were seeded into a 96-well plate at a density of1×10⁶ cells/mL and incubated overnight.

On the following day, the medium was replaced with fresh medium and 5μg/mL of peptide (obtained as described in Experiment example 1) wasadded to the cells. After 30 min incubation of cells with the peptide 50μL of LPS (to a final concentration of 1 μg/mL) was added, and cellswere incubated for additional 24 hr. The experimental sample with theinduction of inflammatory response was treated with 1 μg/mL mLlipopolysaccharide (LPS; Sigma, USA) and control sample was treated withphosphate buffered saline (PBS; pH 7.2). The supernatant samples fromeach condition was collected in eppendorf tubes and subjected to furtheranalysis.

Experiment 2-1. NO Level Analysis

The level of nitric oxide (NO) was measured in Raw 264.7 cell (1×10⁶cell/ml) using Griess reagent system (Promega, USA). Culture medium of50 μl was added to a 96-well plate and Griess reagent I (NED) solutionand Griess reagent II (Sulfanilamide solution) are added in the sameamount. After 10 min incubation of cells with the reagents, the opticaldensity at 540 nm was measured within 30 min using a microplate reader(Molecular Devices, USA). The concentration of NO was calculated byusing a standard curve (0˜100 μM) of sodium nitrite.

As shown in Table 2 below, stimulation of cells with LPS increased theexpression of NO, but in co-treatment with LPS and PEP-1, the expressionlevel of NO mentioned above decreased. NO is produced duringinflammation, and the result showing PEP-1 reduced NO level to 65% ofthe control strongly support the anti-inflammatory effect of PEP-1.

TABLE 2 The measurement of anti-inflammatory effect of human telomerasederived PEP 1 NO Expression Decreased NO Level of Expression Level Testsample control (%) (%) PBS 0 — LPS 1 μg/mL PBS 100 0 PEP 1 (0.5 μg/mL)35 65

Experiment 2-2. Analysis of Cytokine Inhibitory Effect

To investigate the effect of PEP-1 on inhibiting proinflammatorycytokine production RAW 264.7 cell were pre-treated with PEP 1 at aconcentration of 5 μg/mL challenged with LPS at a concentration of 1μg/mL, and cells were further incubated for 24 hr. The supernatantsamples containing cell culture medium was collected and analyzed forthe cytokine levels using ELISA kits (eBioscience, San Diego).

96 wells plates were coated with 100 μL of capture antibodies (dilutedin coating buffer to the concentration recommended by manufacturer'sprotocol) overnight at 4° C. Then, after washing the plates 5 times, 200μL of assay diluents was added to each well and incubated for 1 hr atroom temperature for blocking. After washing each well with wash bufferfive times, cell culture sample or each cytokine standard protein samplewas diluted and 100 μL of each added into each well. The platecontaining samples were incubated overnight at 4° C. Then, after washingthe plate five times with the wash buffer, 100 μL of secondary antibodyconjugated to avidin was added and incubated for 1 hr at roomtemperature.

Following incubation with the secondary antibody, the plate was washedfive times and incubated with 100 μL of avidin-HRP (BD Bioscience) for30 min at room temperature. After washing the plate seven times, 100 μLof TMB solution (Pierce) was added and incubated for 15 min at roomtemperature. The reaction was stopped by adding 50 μl of 2N H₂SO₄ ineach well The optical density at 450 nm was measured using a microplatereader. Statistical analysis was performed by variance analysis usingANOVA procedure of SPSS program, and verified the significance betweenanalyses using Duncan's multiple range test.

Experiment 2-3. IL-6 Secretion Measurement

As shown in Table 3 below, treatment with LPS alone increased thecytokine IL-6 (interleukin-6) secretion. However, co-treatment with LPSand PEP-1 showed a decrease in the level of the proinflammatory cytokineIL-6 secretion. More importantly, after the treatment with PEP-1, thelevel of proinflammatory cytokine secretion decreased by more than 70%,which indicates a robust anti-inflammatory effect of PEP-1.

TABLE 3 Cytokine IL-6 production inhibition by PEP-1 cytokine IL-6production Test sample % of control inhibition % PBS 0 — LPS 1 μg/ml PBS100 0 PEP 1 (5 μg/ml) 28 72

Experiment 2-4. HMGB1, TNF-α, COX-2 Expression Inhibition

Protein expression level was determined by Western blot analysis. Cellsgrown in PEP-1 containing medium were washed with PBS, treated with0.05% trypsin-EDTA, and collected by centrifugation. The collected cellswere dissolved in an appropriate volume of lysis buffer. Intracellulardebris was pelleted by centrifugation, and equal amount of protein fromeach sample was separated by SDS-polyacrylamide gel electrophoresis. Theseparated protein was transferred to nitrocellulose membrane (Schleicherand Schuell, Keene, N.H., USA), then was tested for the antibodyspecific for each protein. The membrane was incubated with ECL (enhancedchemiluminoesence) solution (Amersham Life Science Corp., ArlingtonHeights, Ill., USA), exposed to X-ray, and the level of proteinexpression was analyzed according to the exposure level shown on theX-ray film.

Western blot analysis was performed to determine the inhibitory effectof PEP-1 on the cytokine protein expression. As shown in Table 4 below,stimulation of cells with LPS increased the expression of cytokines;HMGB1, TNF-α and COX. However, if cells were treated with both LPS andPEP-1, the expression level of pro-inflammatory cytokines mentionedabove decreased. The result showing the treatment with PEP-1 decreasedproinflammatory cytokine levels by more than 70% provide strong evidencesupporting the anti-inflammatory effect of PEP-1.

TABLE 4 The measurement of inhibitory effect of PEP-1 onpro-inflammatory cytokine expression level. Cytokine Expression Level(band intensity) % of control Test sample HMGB1 TNF-α COX-2 PBS — — —LPS 1 μg/ml PBS 100 100 100 PEP 1 (5 μg/ml) 30 25 22

Example 3 Investigation of the Inhibitory Effect of PEP-1 on TNF-α Levelin HepG2 Cells

Experiment 3-1: Cell Culture

PBMC (peripheral blood mononuclear cell) was separated from the bloodsamples (50 ml) collected from healthy subjects using Ficoll-Paque™ PLUS(GE Healthcare Life Sciences, Piscataway, N.J., USA). PBMCs were thenenriched in complete RPMI 1640 medium containing 20% of human serum,followed by transferring to 100 mm polystyrene cell culture plate coatedwith human serum for 30 mins. After 2 hr incubation at 37° C. and 5%CO₂, the monocytes were detached from the bottom of cell culture plateusing cold PBS (Phosphate Buffered Saline) (Gibco/Life Technologies,Carlsbad, Calif., USA), and 1×10⁵ cells were cultured in each well of96-well plate in RPMI 1640 medium (supplemented withpenicillin-streptomycin; 100 mg/ml, human serum; 20%) over night.

For Luciferase Analysis, HEK293/null (human embryonic kidney) cells andHEK293/TRL stably expressing TLR2 (toll-like receptor2) obtained fromSeoul National University School of Dental Medicine were used. One daybefore the luciferase experiment, 2.5×10⁵ cells were seeded into eachwell of 12-well plate and cultured overnight in DMEM (Dulbecco'smodified Eagle's medium) medium (supplemented with blasticidin; 10μg/ml, fetal bovine serum; 10%) (Invitrogen/Life Technologies, Carlsbad,Calif., USA)

Experiment 3-2: Cytokine Assay

To see the effect of PEP-1 on TNF-α level in terms of protein expressionlevel, ELISA (enzyme linked immunosorbent assay) was performed. 1×10⁵PBMC-derived monocytes were cultured in 96-well plate over night. Afterthen, LPS (lipopolysaccharide; 10 ng/ml, Sigma) was treated for 2 hours,followed by 3 times washes with PBS. OPTI-MEM medium (Invitrogen/LifeTechnologies, Carlsbad, Calif., USA) was then treated for an hour toinduce the starvation, and 4 μM of FITC (Fluorescein Isothiocyanate),FITC-TAT, PEP-1-FITC, and FITC-PEP-1 were treated for 2 hours beforemeasuring the TNF-α level. After culturing, cell soup was collected, andthe amount of TNF-α was measured using ELISA kit (R&D, Minneapolis,Minn., USA) as follows:

TNF measurement uses sandwich ELISA method. 100 μl of TNF-α primaryantibody was added into each well of pre-coated 96-well plate, and theplate was incubated at 4° C. overnight. On next day, the plate waswashed 3 times with 0.5% Tween 20 wash solution for 5 min each, and then100 μl of each sample and standard solution was added and left at roomtemperature for 2 hrs. After washing the plate like above, 100 μl ofHRP-conjugated secondary antibody was added into each well and left atroom temperature for 2 hrs. Again, plate was washed, and avidin/biotinwas added for measuring the absorbance. TNF-α level of each sample wasquantified using the standard graph calculated from the absorbance ofstandard solution.

PBMC-derived monocytes were stimulated with endotoxin LPS (10 ng/ml) for2 hrs, starved for 1 hr using OPTI-MEM, and then 4 μM of FITC, FITC-TAT,PEP 1-FITC and FITC-PEP 1 were treated for 2 hrs. After incubation,TNF-α level was measured with cell culture medium using ELISA. As aresult, in case of FITC and FITC-TAT, TNF-α level increased due to LPS(6.2 and 6.7 ng/ml, respectively), but TNF-α level significantlydecreased in case of PEP-1-FITC and FITC-PEP-1 (0.17 and 0.25 ng/ml,respectively) and the difference was statistically significant (P<0.01)(FIG. 1).

Experiment 3-3: Luciferase Assay

To investigate the role of PEP 1 in inflammatory response, we evaluatedNF-κB expression patterns through luciferase analysis. First, weincubated HEK293/null and HEK293/TLR2 (Graduate School of Dentistry,Seoul National University) in a 12-well plate for 24 hours, so that wewould get 2.5×10⁵ cells/well. After washing three times with PBS, mediumwas replaced with OPTI-MEM (Invitrogen/Life Technologies, Carlsbad,Calif., USA) and incubated for 4 hours, and then a mixture of 3 μllipofectamine (Invitrogen/Life Technologies), NF-κB luciferase and 10 ngrenilla luciferase (Promega, Madison, Wis., USA) was added into eachwell and again incubated for 4 hours. Lipoprotein pam3cys (long/ml,Sigma-Aldrich, St. Louis, Mo., USA) was put into all of the wells exceptfor those of negative control, and FITC (4 μM) and FITC-PEP 1 (4 μM)were treated for 18 hours before it was washed with PBS for three times.We confirmed the activation of NF-κB through TD-20/20 luminometer(Turner designs, Sunnyvale, Calif., USA) after dissolving (lysis) ofcells by putting 50 μl of passive lysis buffer—provided bydual-luciferase reporter assay system (Promega)—into each well.Transfection efficacy was confirmed by cotransfection of pCMV-renillaluciferase (Promega), and we analyzed results by calibrating theluciferase values.

After transfecting NF-κB luciferase to HEK293/null and HEK293/TLR2 celllines, pam3cys, a synthetic lipoprotein, and FITC (4 μM), a negativecontrol were treated together, and pam3cys with FITC-PEP 1 (4 μM) wereagain treated together to be cultured for 18 hours. The measurement ofNF-κB expression patterns by luciferase strength through lysis of cellswith passive lysis buffer—provided by dual-luciferase reporter assaysystem (Promega)-showed that there was no difference in lipoprotein orFITC-PEP-1 treated or non-treated HEK293/null. However, whenlipoprotein, an agonist of TLR2, was treated to HEK293/TLR2 cell line,NF-κB expression increased (P<0.01) compared to that in untreated,confirming occurrence of inflammatory responses. Also, NF-κB expressionincreased when FITC-PEP 1 was treated together compared to that inuntreated; and expression decreased compared to the negative control inwhich lipoprotein and FITC were treated together (P<0.01) (FIG. 2).Ultimately, we were able to confirm that inflammatory response that canbe caused by TLR 2 is reduced when PEP 1 is treated together.

Experiment 3-4: Reanalysis of Peptides that Affect Levels of Cytokinesin THP1 Cell Line

As a Human acute monocytic leukemia cell line, THP-1 monocyte cell line(American Type Culture Collection (Manassas, Va., USA) was used toreconfirm the effects of PEP 1. Cells were grown at a density of0.5-7×10⁵ cells/mL in RPMI 1640 containing 10% FBS, 0.05 mM2-mercaptoethanol, 100 U/ml penicillin, 100 m/mL streptomycin, andmaintained at 37° C. under 5% CO₂. THP-1 cells were differentiated intomacrophages by treating cells with phorbol myristate acetate (PMA) at100 ng/mL for 24 hr at 37° C. for 24 hr.

All reagents and medium were purchased from Gibco BRL. PMA, LPS and2-mercaptoethanol were purchased from Sigma (St. Louis, Mo., USA).Peptide RIA was synthesized from Peptron (Daej eon, Republic of Korea).Reverse transcription PCR kit was purchased from Promega (Madison, Wis.,USA). RT² SYBR® Green qPCR (qPCR reagents) Mastermix_reagents andQIAZOL® (lysis reagent) were purchased from QIAGEN (Valencia, Calif.,USA).

Following differentiation into macrophages, THP-1 cells were washed twotimes using complete RPMI 1640 (5 min/wash). Then, cells were treatedfor 4 hr. with 10 ng/ml LPS and/or 4 μM peptide MA in FBS free RPMI1640.

Total RNA samples were isolated from peptide-treated THP-1 cells byusing Trizol (QIAZOL® (lysis reagent)) reagent and, and cDNA wassynthesized by reverse transcriptase PCR using reverse transcription PCRkit from Promega following manufacturer's protocol.

Then, real-Time qPCR was performed using CFX96 (Bio-Rad) instrument withSYBR® Green system (qPCR reagents). Primers used in the experiments arefound in Table 5. The PCR cycling conditions were 95° C. for 10 min foractivation of HotStart DNA Taq Polymerase, followed by 45 cycles of 95°C. for 10 sec, 55° C. for 30 sec, and 72° C. for 30 sec. All sampleswere measured in triplicate and differences in gene expression werecalculated using the 2-cycle threshold method. All the data werenormalized against β actin (housekeeping gene) and presented as means of+/−S.E. from at least three independent experiments.

TABLE 5   Primers used for qRT-PCR analysis. SEQ Gene  ID NameDNA sequence 2 tnf- (forward) 5′-CTATCTGGGAGGGGTCTTCC-3′ 3 alpha(reverse) 5′-ATGTTCGTCCTGCTCACAGG-3′ 4 beta(forward) 5′-GGACAAGCTGAGGAAGATGC-3′ 5(reverse) 5′-TCGTTATCCCATGAGTCGAA-3′ 6 il-6(forward) 5′-AAAAGTCCTGATCCAGTTCCTG-3′ 7(reverse) 5′-TGAGTTGTCATGTCCTGCAG-3′ 8 il-8(forward) 5′-GTGCAGTTTTGCCAAGGAGT-3′ 9(reverse) 5′-AATTTCTGTGTTGGCGCAGT-3′ 10 inos(forward) 5′-CACCATCCTGGTGGAACTCT-3′ 11(reverse) 5′-TCCAGGATACCTTGGACCAG-3′ 12 beta (forward) 5′-AGAAAATCTGGCACCACACC-3′ 13 actin(reverse) 5′-GGGGTGTTGAAGGTCTCAAA-3′

As shown in FIG. 3, the cytokines involved in the inflammatory responsesdecreased noticeably by treating with PEP 1.

Example 4 Analysis of Inflammatory Response Induced by Amyloid-β Protein

HMGB1 first undergoes acetylation and translocation to cytoplasm byexternal stimulation. Then it is secreted out of the cell, thereforeserving the role of inflammation-causing cytokine. Because when one hasan inflammation due to such activity, HMGB1 protein is secreted from thecell, and patients with inflammatory diseases such as Churg strausssyndrome, rheumatoid arthritis and Sjogren's syndrome will present withelevated serum levels of HMGB1. Hence, if nucleus contains large amountof HMGB1 even when there is a stimulus that causes inflammation, it issuggestive of the fact that HMGB1 is not being secreted out of the cell,which means inflammation is being suppressed.

Experiment 4-1: Analysis of Survival and Proliferation of Neural StemCells by Anti-Inflammatory effects of PEP-1

First of all, PEP-1 was prepared according to the manufacturing methodsdescribed in Example 1.

Experiment 4-2: Neural Stem Cell Culture and Amyloid-β ToxicityAssessment

After removing the cortex from the head of an embryonic rat that hadbeen pregnant for 13 days, it was cultured for a week with BasicFibroblast Growth Factor (bFGF) to obtain the neural stem cells. Toanalyze the effects of the amyloid-β protein on the neural stem cells,the pre-oligomerized amyloid-β protein of concentrations 0 to 40 μM wastreated on neural stem cells for 48 hours, then CCK-8 assay, BrdU, andTUNEL assay were used for cytotoxicity assessment (refer from BA Yankneret al, 1990 and KN Dahlgren et al, 2002). We used the same concentrationof amyloid-β protein in subsequent experiments after we confirmed thatcell survival was reduced to 60% when processed with 20 μM of amyloid-βprotein (Refer to FIGS. 4 and 5).

Experiment 4-3: Cell Toxicity Assessment by Treatment with PEP-1

To evaluate the impact of PEP-1 on the cultured neural stem cells, theneural stem cells were firstly cultured by a well-known method (BAYankner et al, 1990 and KN Dahlgren et al, 2002). Then, differentconcentrations (0, 1, 10, 50, 100, 200 μM) of PEP-1 were treated for 48hours, followed by cell viability and proliferation assessments usingMTT assay, BrdU and TUNEL assay. PEP-1's concentrations from 0 to 200 μMappeared stable in the neuronal system since they did not inhibit bothsurvival and proliferation of neural stem cells (Refer to FIGS. 6 and7).

Experiment 4-4: Cell Toxicity Assessment by Co-Treatment of Amyloid-βProtein and Telomerase Peptide

To determine whether PEP 1 has the effect of suppressing theneurotoxicity caused by amyloid-β protein, 20 μM amyloid-β protein andvarious concentrations of PEP-1 were co-treated for 48 hours. The cellviability and apoptosis were measured using MMT assay, CCK-8 assay, LDHassay and TUNEL assay, and neural stem cell proliferation by BrdU assay.

The results of MMT assay and CCK-8 assay confirmed that 10 μM of PEP-1began to protect neural stem cells from neurotoxicity by amyloid-β, andthe most effective protection was provided in 100 μM. (Refer to FIG. 8).LDH assay was carried out for assessment of the degree of cell death asanother method, and we confirmed that the increase in cell death byamyloid-β decreased by PEP-1, and efficacy was seen starting at 1 μMconcentration (Refer to FIG. 9).

We also confirmed with BrdU assay that the decreased cell proliferationdue to amyloid-β protein was restored when processed with PEP-1 (Referto FIG. 10).

Cell mobility is a vital matter due to the nature of neural stem cell.According to the experimental results of cell mobility, we confirmedthat the decreased cell proliferation due to amyloid-β protein wasrestored when processed with PEP-1, and that it increased even more whenin 10 μM concentration, compared to control. This suggests that in thefuture clinical trials, processing prior to stem cell transplantationmay draw more effective results. (Refer to FIG. 11).

To confirm the degree of neuronal stem cells damage, TUNEL assay wasperformed. Neuronal stem cell death was observed to be significantlyincreased in 20 μM amyloid-β protein treatment group, and neuronal stemcell death decreased when treated with 1 to 100 μM of PEP 1. (Refer toFIG. 12)

The mechanism of action of PEP-1's protective effect on apoptosis byamyloid-β protein was investigated. First, it was investigated whetherPEP-1 is capable of minimizing the oxidative damage caused by amyloid-βprotein. Change in generation of reactive oxygen species after treatmentwith amyloid-β protein and PEP-1 was observed by using DCF-DA staining(Molecular Probes, Eugene, Oreg.). In the group in which reactive oxygenspecies increased due to 20 μM of amyloid-β protein, the increasedreactive oxygen species decreased by PEP-1 treatment (1 μM, 10 μM, 50μM) (Refer to FIG. 13).

Experiment 4-5: Comparative Analysis of Protein Expression Levelsbetween the Groups Treated With and Without PEP-1

Protein expression level of PEP-1 treated group and untreated group wasanalyzed by 2D-electrophoresis technique and antibody microarraytechnique. Prepared 200 μg by extracting proteome from the neural stemcells cultured in Experiment 3-1 of Example 3. In addition, the group inwhich PEP-1 was not treated was used as the comparison group in the samecondition.

2D-electrophoresis was performed using 12% acrylamide gels. First gelelectrophoresis was performed at PI 4-10N, using a gel size of 8.5×7 cm.After electrophoresis, it was dyed with Colloidal Coomassie Blue, andthen compared expression by using PDQuest software to analyze each spot.

Difference in the expression levels of more than 1.5 times wasidentified using MALDI-TOF MS (Matrix Desoprtion/lionization Time ofFlight Mass Spectromestry). Among these, proteins correlated withinflammation-related signaling, such as i-NOS and HMGB-1 were identified(Refer to Table 6). The changes in protein expression levels eitherincreased or decreased by 1.5 times by amyloid-β protein, but it wasconfirmed that expression level was regulated close to that of negativecontrol when PEP-1 was added (Refer to FIG. 14).

Antibody microarray was carried out by using cell signaling kit (CSAA1,PANORAMA′ Ab Microarray Cell Signaling kit (proteomics tool thatmeasures the relative level of protein expression)), array slides werescanned by GENEPIX® Personal 4100A scanner (Molecular Devices) and thedata were analyzed by GENEPIX® Pro 5.0 (Molecular Devices).

The Table 6 below is an analysis of expression levels of proteinsassociated with inflammation by 2D electrophoresis technique. Thecontrol group represents protein expression level of cells that were nottreated with neither amyloid-β protein nor PEP-1. It shows increased ordecreased multiple of protein expression based on the control group'sexpression level.

We confirmed with the results of analysis that like the suggested inTable 6 below, inflammation related protein over-expression orunder-expression was controlled by PEP-1; the protein expression levelwas close to that of negative control group.

TABLE 6 20 μg 20 μg β-amyloid β-amyloid + PEP 1 Negative treated grouptreated group Protein Control (fold) (fold) HSP 70 1.0 −2.3 1.2 HSP 901.0 −1.8 1.0 HMGB1 1.0 −1.5 2.8 GADD 153 1.0 1.6 1.2 i-NOS 1.0 1.9 −1.1e-NOS 1.0 1.9 −1.1 Pyk2 1.0 2.0 1.2 MAP Kinase 1.0 2.2 1.0

Phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway serves acrucial role in the growth and survival of neuronal stem cells. PI3Kpathway is activated by growth factors and regulatory factors, and isinvolved in the normal regulation of neuronal stem cell growth andsurvival. AKT signaling pathways disable several pro-apoptotic factors,including a well-known apoptotic signaling molecule, GSK3β.

To further investigate the anti-inflammatory effects of PEP-1, weperformed Western blot on HMGB1, since it showed a major change inprotein analysis. As a result, the processing of the PEP-1 increased theprotein expression levels in anti-apoptotic proteins such as Ki67, pAKT,PI3K, HSTF-1 and Bcl-2, and decreased the protein expression levels ofapoptotic signals such as Bax, GSK3β, Cytochrom-c, caspase-3 (Refer toFIG. 15).

HMGB1, a non-histone structure protein that binds to DNA, serves diverseroles within a cell; such as stabilizing nucleosome structure andregulating gene expression. As one of the inflammation-causing substancethat is excreted in the late phase of inflammatory response, it isexcreted by macrophages and monocytes when inflammation is stimulated,but when neuron is significantly damaged and leads to cell necrosis, itwill be excreted out of the cell, causing an intense inflammatoryresponse. The increase of HMGB1 by PEP-1 treatment after the decrease byamyloid-β treatment in the cytoplasm of the nerve cells reflects thefact that PEP-1 inhibits secretion of HMGB1 out of the cell caused byneuron cell death; therefore suggesting that PEP-1 has powerfulanti-inflammatory effects (Refer to FIG. 15).

In addition, we investigated the response of PEP-1 to the amyloid-βaggregation. Aggregation of protein was inhibited when treated withPEP-1 (Refer to FIG. 16(A)) in induction of aggregation of amyloid-β,and protein underwent degradation when PEP-1 was treated on theamyloid-β protein that was already induced for aggregation (Refer toFIG. 16(B)).

In the mechanism of action of PEP-1, we have previously confirmed theincrease in cell survival signaling and decrease in apoptosis signalingof PI3K. To investigate whether these effects are direct or indirect, wetreated PI3K-inhibitor, LY294002 (Promega). As a result, the increasedcell viability after treating with PEP 1 decreased when treated withLY294002. Thus, we can conclude that PI3K is directly associated withPEP 1's neuroprotective effect (Refer to FIG. 17).

PEP-1 inhibits apoptosis of neural stem cells by amyloid-β protein.Also, the improvement of cell mobility of neural stem cells wasconfirmed, therefore suggesting a variety of possibilities in clinicalapplication. The inhibition effects from neurotoxicity caused bybeta-amyloid protein was verified by the anti-inflammatory effect of themechanism of action of PEP 1, increased survival factors of neuro stemcells and decreased apoptotic factors, especially activation of PI3Ksignaling pathway and antioxidant effects.

Example 5

qPCR array

Materials and Methods

THP-1 Cell Culture

THP-1 cells (human monocytic leukemia derived cell line) were purchasedfrom ATCC (American Type Culture Collection, Manassas, Va., USA) andcultured in RPMI-1640 (Life technologies, Carlsbad, Calif., USA) mediumsupplemented with 10% FBS (Life technologies), 1%penicillin/streptomycin (Life technologies), and 0.05 mM2-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo., USA) at 37° C. in 5%CO₂. THP-1 cells that normally grow in suspension were differentiatedinto an adherent macrophage-like phenotype in differentiation medium(complete growth medium containing 100 ng/mL phorbol 12-myristate13-acetate (PMA, Sigma-Aldrich)) for 24 hr. For differentiation, THP-1cells (3×10⁶ cells/plate, ˜95% confluency) were seeded into 10-cm tissueculture plates and incubated in differentiation medium.

Treatment of THP-1 Cells with Anti-Inflammatory Peptide, PEP-1

Following differentiation, the macrophage-like THP-1 cells were washedtwice using complete growth medium. Then, cells were treated with 10ng/mL lipopolysaccharide (Sigma-Aldrich) and/or 4 μM PEP-1 for 4 h at37° C.

RNA Isolation and cDNA Synthesis from THP-1 Cells

Total RNA was extracted and purified using RNeasy mini kit from Qiagen(Valencia, Calif., USA) following manufacturer's protocol. cDNAs weresynthesized by reverse transcription using Reverse Transcription Systemfrom Promega (Madison, Wis., USA) according to the manufacturer'sprotocol.

PCR Arrays

Then, cDNA samples from THP-1 cells were used as template for real-timequantitative PCR (qPCR) analysis. For qPCR analysis, RT² Profiler PCRArray kits were purchased from SABiosciences/Qiagen (Valencia, Calif.,USA). Four different PCR array kits analyzing separate signalingpathways used in the experiment are as follows: human signaltransduction pathway finder, human inflammatory cytokines & receptors,human transcription factors, human NF-κB signaling pathway. PCR wasperformed with SYBR® Green qPCR system (qPCR reagents, Qiagen) using aBio-Rad (Hercules, Calif., USA) CFX 96 real-time PCR instrument.Thermocycling conditions were: 95° C. for 10 sec; 55° C. for 30 sec; 95°C. for 10 min; 95° C. for 10 sec, 55° C. for 30 sec, and 72° C. for 30sec for 50 amplification cycles. Data represents the average value fromthree independent experiments, and % decrease was determined by thetarget gene expression in LPS treated samples vs. LPS+PEP-1 treatedsamples. Among 336 genes analyzed, only those showing statisticallysignificant (p<0.05, student's t-test) % decrease were shown in table 7.

Results

TABLE 7 PEP-1 inhibits the genes shown in the table above (*, NF-κBtarget genes containing consensus NF-κB binding sites in the promoterregion). % Decrease by PEP 1 (LPS + PEP-1/LPS), Signalling pathway Genep < 0.05 TNFα* ↓↓34% IL10* ↓↓32% IL1Ra* ↓20% IL17C ↓↓35% G-CSF* ↓↓↓48%GM-CSF* ↓↓29% CCL4/MIP1β* ↓22% CCL26/MIP4α ↓↓↓42% TNF Receptor FamilyTNFR11β ↓↓↓38% CD40Ligand ↓18% Lipid biosynthesis ACSL5 (acyl-coAsynthase) ↓17% Apoptosis BCL10 ↓↓32% NFκB IκBα ↓↓27%

PEP-1 inhibited transcription of genes shown in Table 7 with THE %inhibition calculated as the ratio in the level of transcription betweenLPS treated vs. LPS+PEP-1-treated samples (THP-1 cells). Among the 336genes analyzed, only the 13 genes in Table 7 showed statisticallysignificant decrease following PEP-1 treatment. Those genes can begrouped into different functional categories, which include chemokines &cytokines, TNFα receptor signaling, lipid metabolism, apoptosis, andNF-κB signaling. More importantly, genes in the chemokines & cytokinescategory have been known as NF-κB target genes, having NF-κB consensusDNA-binding sites in their promoter regions. Taken together, data fromPCR arrays support that PEP-1 may exert anti-inflammatory effects bymodulating the master regulator of inflammation NF-κB, and by doing soPEP-1 can be used as anti-inflammatory therapeutic agents in a widerange of inflammatory diseases.

The invention claimed is:
 1. A method for treating a urogenital disordercomprising administering to a subject in need thereof a compositioncomprising the isolated peptide of SEQ ID NO:
 1. 2. The method accordingto claim 1, wherein the inflammatory disease urogenital disorder isselected from the group consisting of epididymitis, vaginitis,prostatitis, and urethritis.
 3. The method of claim 1, wherein thecomposition is administered through oral, rectal, transdermal,intravenous, intramuscular, intraperitoneal, in the bone marrow,epidural, or subcutaneous means.
 4. The method of claim 1, wherein thecomposition comprises 0.1 μg/mg to 1 mg/mg of the isolated peptide. 5.The method according to claim 1, wherein the peptide is administered ina single dose at a concentration of 0.1 μg/kg to 1.0 g/kg.
 6. The methodaccording to claim 1, wherein the peptide is administered in a singledose at a concentration of 1 μg/kg to 10 mg/kg.
 7. The method accordingto claim 1, wherein the peptide is administered 1 to 3 times a day. 8.The method of claim 1, wherein the peptide is administered at a dailydose of 0.1 μg/kg to 1.0 g/kg.
 9. The method of claim 8, wherein thepeptide is administered 1 to 3 times daily.